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A single-molecule long-read survey of the human transcriptome
Nature Biotechnologyvolume 31, pages1009–1014 (2013)Cite this article
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Abstract
Global RNA studies have become central to understanding biological processes, but methods such as microarrays and short-read sequencing are unable to describe an entire RNA molecule from 5′ to 3′ end. Here we use single-molecule long-read sequencing technology from Pacific Biosciences to sequence the polyadenylated RNA complement of a pooled set of 20 human organs and tissues without the need for fragmentation or amplification. We show that full-length RNA molecules of up to 1.5 kb can readily be monitored with little sequence loss at the 5′ ends. For longer RNA molecules more 5′ nucleotides are missing, but complete intron structures are often preserved. In total, we identify∼14,000 spliced GENCODE genes. High-confidence mappings are consistent with GENCODE annotations, but >10% of the alignments represent intron structures that were not previously annotated. As a group, transcripts mapping to unannotated regions have features of long, noncoding RNAs. Our results show the feasibility of deep sequencing full-length RNA from complex eukaryotic transcriptomes on a single-molecule level.
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Acknowledgements
We thank J. Eid and L. Hickey at Pacific Biosciences for providing alignment statistics of reads to reference genomes. We thank J. Kelley, C. Araya, D. Phanstiel, S. Shringarpure and M. Sikora at Stanford as well as J. Korlach at Pacific Biosciences for comments on this manuscript. We would like to also thank T. Daley and A. Smith at USC for advice on modeling library complexity. This work was supported by US National Institutes of Health grants 5P01GM099130-02, 5U54HG00699602-02 and 5U01HL107393-03, and by the US National Institues of Health training grant 5 T32 HD07149.
Author information
Donald Sharon and Hagen Tilgner: These authors contributed equally to this work.
Authors and Affiliations
Department of Genetics, Stanford University, Stanford, California, USA
Donald Sharon, Hagen Tilgner, Fabian Grubert & Michael Snyder
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA
Donald Sharon
- Donald Sharon
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- Hagen Tilgner
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- Fabian Grubert
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- Michael Snyder
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Contributions
All authors proposed the project. D.S. devised and performed experiments and wrote the first version of the introduction. H.T. devised and performed analysis, prepared figures and wrote the first version of results and discussion. All authors discussed experiments and analysis and collaborated on the final version.
Corresponding author
Correspondence toMichael Snyder.
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Competing interests
M.S. is on the scientific advisory board of Personalis and GenapSys. All other authors declare no competing interests.
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Sharon, D., Tilgner, H., Grubert, F.et al. A single-molecule long-read survey of the human transcriptome.Nat Biotechnol31, 1009–1014 (2013). https://doi.org/10.1038/nbt.2705
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